Spores are known to form from aerobic Bacilli, anaerobic Clostridia, selected sarcinae and a few actinomycetes. Spores resemble certain plant seeds in that they do not carry out any metabolic reactions. In this regard they are especially suited to withstand severe environmental stress and are known to survive prolonged exposures to heat, drying, radiation and toxic chemicals. These properties make spores especially difficult to kill in environments, like living tissue or objects which come in contact with living tissue, which would be adversely effected by extreme conditions.
Fungi, viruses and vegetative cells of pathogenic bacteria are sterilized within minutes at 70 degrees centigrade; many spores are sterilized at 100 degrees centigrade. However, the spores of some saprophytes can survive boiling for hours. Heat is presently the most commonly used means to insure sterilization of spores.
The outer coat of spores is made of a keratin-like protein which comprises as much as 80% of the total protein of the spore. It is this protein coat which is responsible for the resistance of spores to chemical sterilizing agents. A variety of compounds have been used to insure sterilization of spores and have found acceptance depending upon constraints imposed by environment and the required efficacy of action. Acids, alkali, phenols, iodophors, salts, heavy metals, chlorine, hypochlorite, alcohols, glutaraldehyde, formaldehyde, ethylene oxide, organic solvents and surfactants all have been shown to have some action as a sterilant. However, of these compounds only aldehydes, ethylene oxide, hypochlorites, and Alcide (EPA Reg. No. 456310-03), are commonly used commercially to kill spores.
Iodine and tincture of iodine, a 2-7% solution of iodine in aqueous alcohol containing KI is known to kill bacteria but not known to be generally effective against spores. Additionally, this mixture has a painful and destructive effect on living tissue. Hydrogen peroxide has been and is widely used as an antiseptic but bacteria and spores vary widely in their susceptibility; spores in particular require high concentrations of hydrogen peroxide (3% and up) and long periods (hours, days) of contact with hydrogen peroxide to insure good killing efficacy.
Hydrogen peroxide is known to dissociate into free radicals. The rate at which free radical species are generated from the decomposition of hydrogen peroxide is believed in accordance with the present invention to determine the sporocidal efficacy of this compound. Enzyme catalyzed reactions are known to occur 10 to 15 orders of magnitude more rapidly than the corresponding non-enzymatic reactions. In accordance with the present invention an enzyme, peroxidase, has been selected to catalyze the reduction of hydrogen peroxide for generating free radicals from iodide which has been added to an aqueous based medium in the form of one of its salts.
The enzyme peroxidase catalyzes the transfer of electrons from iodide to hydrogen peroxide. When an electron is removed from an iodide anion this molecule is transformed into a free radical; this free radical or a by-product of this molecule is the sporocidal entity. The free radicals generated in this process are generated at greatly elevated rates relative to the rate at which free radicals are generated from the non-enzymatic dissociation of peroxide.
Peroxidases are classified as enzymes which act to reduce hydrogen peroxide. The different types of peroxidases are distinguished by the donor molecules they use; donor molecules supply electrons which peroxidase donates to hydrogen peroxide. In accordance with the present invention a peroxidase is used to generate free radicals from iodide. Iodide is capable of acting as a substrate for horseradish peroxidase in the reduction of hydrogen peroxide.
The method of the present invention teaches a practical means to control the generation of free radical species to form a sporocide having a defined period of sporocidal activity. The sporocide of the present invention is formed by combining three components, viz., a peroxide or peroxide generating material, a peroxidase and a source of iodide . . . i.e. an iodide salt. The in-situ sporocidal disinfectant will continuously generate free radicals over a defined period of time depending upon the concentration level of each component in the sporocide.
The duration of peroxide reduction and the amount of sporocidal entities produced can be controlled by careful formulation of the three components comprising the system. As long as the enzymatic reduction of hydrogen peroxide continues, free radicals will be generated. The free radicals being generated have an extremely short lifetime and as such must be continuously generated to prolong the period of sporocidal activity. The duration of the reaction, and therefore its sporocidal lifetime, is controlled via the formulation. Other factors remaining constant, the longer the reaction occurs the greater the sporocidal effectiveness.
The method of the present invention teaches how to maintain the sporocide in a nonreacting state; how to activate the sporocide at the critical moment when sterilization is desired and how to control the generation of free radicals over a preselected time period to complete the sterilization.
The selection of donor molecule is critical to the invention. The use of iodide salts in the sporocide is of paramount importance since the iodide molecules or products thereof are the molecules transformed in the reaction into the sporocidal agents. The instantaneous dissolution rate and very high solubility of iodide salts like sodium iodide and potassium iodide make them ideal candidates for this application. Many salts of iodides may be used either alone or in combination with other iodides. The choice will usually be dictated by cost and manufacturability of the materials chosen.
Any aqueous based medium which does not interfere with the peroxidative catalytic cycle and does not introduce compounds which are capable of reacting with the products or by-products of the peroxidative catalytic cycle is suitable for the practice of this technology. That is, the medium should allow for the catalytic reduction of hydrogen peroxide and the formation of the sporocidal entities normally found in simple buffered solutions like 0.010 molar sodium phosphate, pH 7.0. From the following examples, it will be apparent that the method of the present invention reduces the concentration of spores in solution by log order magnitudes to a final concentration where the count is essentially zero. Accordingly, contacting spores with a sporicide in solution formed in accordance with the present invention will result in sterilizing the solution substantially free of spores.
The present invention incorporates a peroxide or a peroxide generating compound, a peroxidase, and a salt of iodide like sodium or potassium iodide, which are readily available, inexpensive and easily incorporated into a powder or pill format.